Opportunistic infections caused by Cryptosporidium parvum and Toxoplasma gondii represent life threatening diseases for immuno-compromised patients, children and the elderly. There are currently no effective treatments available for cryptosporidiosis and treatments for toxoplasmosis require the coadministration of sulfadioxine, a compound to which many patients have severe adverse reactions. The development of novel therapeutics that are highly potent and highly selective for the pathogen is of immediate importance. Crystal structures of the validated drug target, dihydrofolate reductase-thymidylate synthase (DHFR-TS), a bifunctional enzyme in protozoa, from C. parvum and T. gondii, provide essential evidence for structure-based drug design against these targets. In the first Aim of this proposal, we will design trimethoprim analogs to interact favorably with species-selective elements of the C. parvum DHFRTS structure. Species-selective elements will be determined by comparing crystal structures of pathogenic DHFR-TS and human DHFR. New inhibitors will be modeled into the structure of the enzyme, while accounting for ligand-induced conformational changes, and binding modes predicted. Crystal structures of trimethoprim analogs will guide future design in an iterative cycle. In the second Aim, we will improve the potency and selectivity of a promising T. gondii DHFR-TS inhibitor, using information from crystal structures of T. gondii DHFR-TS bound to lipophilic inhibitors. Designs for the improvement of T. gondii DHFR-TS inhibitors will take advantage of species-selective elements. In the third Aim, we will elucidate the structural basis of pyrimethamine resistance in DHFR and design potency and selectivity into a novel inhibitor of pyrimethamine-resistant T. gondii DHFR-TS. DHFR resistance to pyrimethamine and other antimicrobials is a threatening problem and novel therapeutics capable of inhibiting the resistant enzymes are desperately needed. We will solve a crystal structure of pyrimethamine-resistant DHFR-TS bound to a novel and exciting inhibitor that shows an inhibition constant of 350 nM against the resistant enzyme in preliminary studies. Using the structural information we will elucidate the structural basis of resistance and modify the novel inhibitor for greater potency and selectivity.